Thermally-tolerant pectin methylesterase

Abstract

Enzymes accumulated in plant cell walls serve diverse physiological functions including metabolism, polysaccharide structure modification, and molecular communication in interactions with other organisms. Pectin methylesterases are economically important enzymes for their impact on quality and processing properties of fruit and vegetable food products. We have now purified TT-PME to homogeneity from sweet orange finisher pulp and determined the complete corresponding nucleic acid sequence. Purified TT-PME was observed by SDS-PAGE as two doublet bands with molecular masses of approximately 46,000 Da and 56,000 Da. Direct Edman sequencing from these proteins showed a common N-terminal peptide. De novo sequencing of eight TT-PME tryptic peptides determined by MALDI-TOF/TOF mass spectrometry provided additional internal sequences. TT-PME did not correspond to any previously reported Citrus spp. PME sequence. Our results show Citrus TT-PME is a distinctive new isoform with phylogenetic relationship closer to PME isoforms in other species rather than to previously described Citrus PME genes.

1. An isolated or recombinant nucleic acid molecule wherein said nucleic acid molecule encodes a pectin methylesterase polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, said polypeptide having stability under harsh reaction conditions comprising a pH range of about 3.5 to about 9.5 and a temperature range from about 45° C. up to about 85° C. 
     
     
       2. The isolated or recombinant nucleic acid molecule according to  claim 1 , wherein said nucleic acid molecule comprises a nucleotide sequence identified by SEQ ID NO: 1. 
     
     
       3. An isolated or recombinant nucleic acid molecule, wherein said nucleic acid molecule comprises a nucleotide sequence that comprises the complement of any one of the nucleotide sequences of  claim 1  or  2 . 
     
     
       4. The isolated or recombinant nucleic acid molecule of any one of  claim 1  or  2 , further comprising one or more regulatory elements operatively linked to said nucleic acid. 
     
     
       5. A DNA construct comprising the nucleic acid molecule of  claim 1 , wherein said nucleotide sequence is linked to a promoter that drives expression in a host cell. 
     
     
       6. A DNA construct comprising the nucleic acid molecule of  claim 2 , wherein said nucleotide sequence is linked to a promoter that drives expression in a host cell. 
     
     
       7. A vector comprising the DNA construct of  claim 5 . 
     
     
       8. A host cell comprising the vector of  claim 7 . 
     
     
       9. The host cell of  claim 8 , wherein said host cell is a single-celled or lower or higher multi-celled organism into which the construct according to the invention can be introduced wherein expression of said construct results in the production of pectin methylesterase polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2.